Pharmaceutical preparation

ABSTRACT

The present invention relates to a pharmaceutical preparation for treating an inflammatory condition, preferably a condition associated with ischemia comprising: a) a physiological solution comprising peripheral blood mononuclear cells (PBM-Cs) or a subset thereof, or b) a supernatant of the solution a), wherein the solution a) is obtainable by cultivating PBMCs or a subset thereof in a physiological solution free of PBMC-proliferating and PBMC-activating substances for at least 1 h.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/140,120 filed Jun. 16, 2011, now U.S. Pat. No. 10,478,456, which is anational phase application of PCT/EP2009/067536, filed Dec. 18, 2009,which claims priority to European Patent Application No. 08450199.8filed Dec. 18, 2008, the disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION Sequence Listing

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 24, 2020, isnamed “16785-194-1_2020-02-24_Sequence-Listing_ST25” and is 4.00 kb insize.

DESCRIPTION OF RELATED ART

The present invention relates to a pharmaceutical preparation fortreating internal inflammatory conditions, preferably internalconditions associated with ischemia.

Hypoxia, a state of reduced oxygen, can occur when the lungs arecompromised or blood flow is reduced. Ischemia, reduction in blood flow,can be caused by the obstruction of an artery or vein by a blood clot(thrombus) or by any foreign circulating matter (embolus) or by avascular disorder such as atherosclerosis. Reduction in blood flow canhave a sudden onset and short duration (acute ischemia) or can have aslow onset with long duration or frequent recurrence (chronic ischemia).Acute ischemia is often associated with regional, irreversible tissuenecrosis (an infarct), whereas chronic ischemia is usually associatedwith transient hypoxic tissue injury. If the decrease in perfusion isprolonged or severe, however, chronic ischemia can also be associatedwith an infarct. Infarctions commonly occur in the spleen, kidney,lungs, brain and heart, producing disorders such as intestinalinfarction, pulmonary infarction, ischemic stroke and myocardialinfarction.

Pathologic changes in ischemic disorders depend on the duration andseverity of ischemia, and on the length of patient survival. Necrosiscan be seen within the infarct in the first 24 h and an acuteinflammatory response develops in the viable tissue adjacent to theinfarct with leukocytes migrating into the area of dead tissue. Oversucceeding days, there is a gradual breakdown and removal of cellswithin the infarct by phagocytosis and replacement with a collagenous orglial scar.

Hypoperfusion or infarction in one organ often affects other organs. Forexample, ischemia of the lung, caused by, for example, a pulmonaryembolism, not only affects the lung, but also puts the heart and otherorgans, such as the brain, under hypoxic stress. Myocardial infarction,which often involves coronary artery blockage due to thrombosis,arterial wall vasospasms, or viral infection of the heart, can lead tocongestive heart failure and systemic hypotension. Secondarycomplications such as global ischemic encephalopathy can develop if thecardiac arrest is prolonged with continued hypoperfusion. Cerebralischemia, most commonly caused by vascular occlusion due toatherosclerosis, can range in severity from transient ischemic attacks(TIAs) to cerebral infarction or stroke. While the symptoms of TIAs aretemporary and reversible, TIAs tend to recur and are often followed by astroke.

Occlusive arterial disease includes coronary artery disease, which canlead to myocardial infarction, and peripheral arterial disease, whichcan affect the abdominal aorta, its major branches, and arteries of thelegs. Peripheral arterial disease includes Buerger's disease, Raynaud'sdisease, and acrocyanosis. Although peripheral arterial disease iscommonly caused by atherosclerosis, other major causes include, e.g.,diabetes, etc. Complications associated with peripheral arterial diseaseinclude severe leg cramps, angina, abnormal heart rhythms, heartfailure, heart attack, stroke and kidney failure.

Ischemic and hypoxic disorders are a major cause of morbidity andmortality. Cardiovascular diseases are responsible for 30% of deathsworldwide. Among the various cardiovascular diseases, ischemic heartdisease and cerebrovascular diseases cause approximately 17% of deaths.

Currently, treatment of ischemic and hypoxic disorders is focused onrelief of symptoms and treatment of causative disorders. For example,treatments for myocardial infarction include nitroglycerin andanalgesics to control pain and relieve the workload of the heart. Othermedications, including digoxin, diuretics, amrinone, beta-blockers,lipid-lowering agents and angiotensin-converting enzyme inhibitors, areused to stabilize the condition, but none of these therapies directlyaddress the tissue damage produced by the ischemia and hypoxia.

Due to deficiencies in current treatments, there remains a need formethods that are effective in treating conditions involving hypoxia.There is also a need for methods that are effective in the prevention oftissue damage caused by ischemia that occurs due to, e.g.,atherosclerosis, diabetes and pulmonary disorders.

Conditions associated with ischemia and hypoxia are usually accompaniedby inflammation. Therefore, means and methods are needed which alsoreduce inflammation.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide means which allowthe efficient treatment of internal inflammatory conditions, preferablyconditions associated with ischemia.

The present invention relates to a pharmaceutical preparation fortreating an internal inflammatory condition, preferably an internalcondition associated with ischemia, comprising

a) a physiological solution comprising peripheral blood mononuclearcells (PBMCs), which consist of lymphocytes (T cells, B cells, NK cells)and monocytes, or a subset thereof, or

b) a supernatant of the solution a), wherein the solution a) isobtainable by cultivating PBMCs or a subset thereof in a physiologicalsolution free of PBMC-proliferating and PBMC-activating substances forat least 1 h.

It turned out that the administration of a pharmaceutical preparation asdefined above to a patient suffering from an internal inflammatorycondition, preferably an internal condition associated with ischemia,results in an alleviation of the respective symptoms and in a healingprocess.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a flowchart of Induction of MCI.

FIG. 1B is a graph of the percentage of irradiated and non-irradiatedrat PBMCs positively stained for Annexin after a culture period of 18 h.

FIG. 2A is a FACS plot of irradiation and apoptosis.

FIG. 2B is a graph of co-incubation of LPS stimulated PBMCs or monocyteswith irradiated apoptotic autologous PBMCs.

FIG. 2C is a graph of IL-6 secretion profile of LPS stimulated PBMCs andmonocytes in the presence of IA-PBMCs.

FIG. 2D is a graph of autologous IA-PBMCs in a mixed lymphocyte reactionwith LPS stimulation.

FIG. 2E is a graph of RT-PCR RNA expression of VEGF, IL-8/CXCL8, and MMPtranscripts.

FIG. 2F is a graph of ELISA VEGF, IL-8/CXCL8, and MMP9.

FIG. 2G is a graph of human fibroblasts incubated in supernatants.

FIGS. 3A, 3B, and 3C show CFSE labeled syngeneic PBMCs administered viathe tail vein in rats after artificial myocardial infarction. Anexemplary field of view of CFSE+ cells isolated from each of the liver(FIG. 3A), the spleen (FIG. 3B), and the heart (FIG. 3C) is shown aboveits corresponding graph illustrating the quantified CFSE+ cells/HPFobserved. As shown, CFSE labeled syngeneic PBMCs were predominantlyfound in the spleen (FIG. 3B), to a lesser extent in the liver (FIG.3A), and no cells in the infarcted heart (FIG. 3C).

FIGS. 3D, 3E, and 3F show HE stained infarct zones of rats injected witheither medium.

FIGS. 3G, 3H, and 3I show the results from rats treated with viablecells with an exemplary field of view of CD68+ cells/HPF illustratedabove each respective quantification of CD68+ cells/HFP.

FIGS. 3J, 3K, and 3L show exemplary fields of view of S100β+ cells/HPFillustrated above each respective quantification of S100β+ cells/HPF. Asshown, higher levels of S100β+ cells were found in rats receiving mediumalone compared to the application of viable PBMCs or IA-PBMCs.

FIGS. 4A, 4B, and 4C graphs amounts of cells staining positive for VEGFthat were detected in infarcted myocardial tissue obtained from animalsinjected with IA-PCMC (FIG. 4C), in comparison with medium (FIG. 4A), orviable cell treatment (FIG. 4B). Exemplary fields of view of VEGF+cells/HPF are illustrated above each respective quantification.

FIGS. 4D, 4E, and 4F show expression patterns for VEGF receptor KDR/FLK1with exemplary fields of view for FLK1+ cells/HPF illustrated above eachrespective quantification.

FIGS. 4G, 4H, and 4I show detected CD34 in all three groups (FIGS. 4D,4E, and 4F, respectively) with exemplary fields of view for cD34+cells/HPF illustrated above each respective quantification.

FIGS. 4J, 4K, and 4L show immunohistological analysis for the markerc-kit with exemplary fields of view for c-kit+ cells/HPF illustratedabove each respective quantification.

FIGS. 5A, 5B, and 5C show histological analysis of ischemic rat heartsexplanted 6 weeks after induction of myocardial infarction.

FIG. 5D is a statistical analysis of data obtained from planimetricanalysis of specimen collected 6 weeks after LAD-ligation.

FIGS. 5E, 5F, and 5G show assessment of cardiac function parametersshortening fraction, ejection fraction, and endsystolic diameter.

FIG. 6A is a graph of showing that neither unstimulated viable PBMCs orIA-PBMCs secrete the mainly monocyte derived pro-inflammatory cytokineTNF-α.

FIG. 6B is a graph showing a strong induction of pro-inflammatoryInterferon-γ secretion after activation as compared to unstimulatedPBMCs.

FIG. 7A is a graph of pooled results of flow cytometric analysis.

FIG. 7B displays a representative FACS analysis of PBMCs eitheractivated (PHA, CD3 mAb). Gating represents % of positive cells.

FIG. 8 shows high proliferation rates as measured by 3[H]-thymidineincorporation of stimulated PBMCs when compared to viable PBMCs culturedin RPMI without stimulation.

FIG. 9 shows inhibition of T cell response of PBMC secretoma in T cellproliferation assays.

FIGS. 10A-10F show supernatant levels of Interleukin-8, Gro-alpha,ENA-78, ICAM-1, VEGF and Interleukin-16.

FIG. 11 shows the extension of myocardial scar tissue 6 weeks afterexperimental LAD ligation (as % of the left ventricle).

FIGS. 12A, 12B, and 12C show macroscopic appearance of rat heartsexplanted 6 weeks after experimental myocardial infarction.

FIGS. 13A-13D show representative echocardiographic analyses (M-Mode).

FIGS. 14A and 14B show echocardiographic analyses conducted 6 weeksafter myocardial infarction.

FIG. 15 shows the Kaplan-Meier survival curve for all four treatmentgroups.

FIG. 16 shows anti-CD3 and PHA stimulation experiments performed withPBMCs.

FIGS. 17A-17C show the proliferation of PBMCs upon stimulation withanti-CD3 (FIG. 17A), PHA (FIG. 17B), and mixed lymphocytes (FIG. 17C).

FIGS. 18A and 18B show the level of Annexin V (FIG. 18A) and PIpositivity (FIG. 18B) of the supernatant of CD4+ cells incubated withPBMC supernatants.

FIGS. 19A and 19B show the inhibition of the up-regulation of CD25 (FIG.19A) and CD69 (FIG. 19B) in CD4+ cells by PBMC supernatant.

FIG. 20 shows that the demonetizing of IL-10 and TGF-β did not increasethe proliferation rates of CD4+ cells.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical preparation of the present invention comprisescultivated PBMCs or a subset thereof and/or the supernatant in which thePBMCs have been cultivated. In the course of the cultivation of PBMCsthese cells express and secrete substances like cytokines which differfrom those expressed and secreted in activated PBMCs. This means thatthe secretome of PBMCs of the present invention is different from thesecretome of activated PBMCs. The cells of the present invention undergoa non-cell-surface moiety triggered secretome production. Therefore itis surprising that PBMCs which have not been contacted with PBMCactivating substances like PHA or LPS can be employed to treat internalinflammatory conditions, in particular ischemic conditions, which showsthat the secretome of these cells comprises substances supporting thetreatment of such or similar conditions.

The PBMCs according to the present invention are obtainable bycultivation in a physiological solution which does not comprisePBMC-proliferating and PBMC-activating substances. However, the PBMCsare incubated in the physiological solution for at least 1 h. Thisminimum time of cultivation is required to let the PBMCs secretecytokines and other beneficial substances.

PBMCs part of the preparation according to the present invention can beobtained from whole blood using methods known in the art such as Ficollgradient, hypotonic lysis etc. These methods are well known in the art.

PBMCs of the pharmaceutical preparation may be obtained from a pool ofdonors or from the same individual to which the preparation will beadministered.

The PBMCs or the subsets thereof are present in the preparationaccording to the present invention in their viable form.

The physiological solution from which the supernatant is obtainedcomprises at least 500, preferably at least 1000, more preferably atleast 10⁵, even more preferably at least 10⁶, cells per mL solution orper dosage unit.

The preparation of the present invention may comprise at least 500,preferably at least 1000, more preferably at least 10⁵, even morepreferably at least 10⁶, PBMCs per mL or per dosage unit.

“Physiological solution”, as used herein, refers to a liquid solution inwhich PBMCs are cultivated prior to their use in the pharmaceuticalpreparation according to the present invention.

“Physiological solution” refers also to a solution which does not leadto the death of PBMCs within an hour, preferably within 30 min. If thenumber of viable PBMCs is decreasing in a solution by 75%, morepreferably by 90% within one hour, preferably within 30 min, thesolution is not considered to be a “physiological solution” as definedherein. The “physiological solution” does not lead to a spontaneouslysis of PBMCs when contacted with said solution.

In this context the step of “cultivating” or “culturing” comprises orconsists of the step of “incubating”, a step in which the cells arecontacted with a solution for a defined time (at least 1 h, preferablyat least 4 h, more preferably at least 8 h, even more preferably atleast 12 h) under conditions which are regularly used for cultivatingPBMCs.

The term “condition associated with ischemia” in the context of thepresent invention can be used interchangeably with the term “ischemicconditions” and denotes any condition, disease or disorder in whichregions of the human or animal body are deprived of adequate oxygensupply resultant damage or dysfunction of tissue. A pathologicalcondition may be characterized by reduction or abolition of blood supplywithin an organ or part of an organ, which may be caused by theconstriction or obstruction of a blood vessel. Such conditions arecollectively referred to herein by the term “ischemia” or “ischemiarelated conditions” or “condition related to ischemia”. In heartdisease, for instance, ischemia is often used to describe the heartmuscle that is not getting the proper amount of oxygen-rich bloodbecause of narrowed or blocked coronary arteries. The symptoms ofischemia depend on the organ that is “ischemic”. With the heart,ischemia often results in angina pectoris. In the brain, ischemia canresult in a stroke. Ischemia conditions are accompanied by inflammation.

Non-limiting examples for pathological conditions which relate toinflammation, in particular to ischemia, include wounds, myocardialischemia, limb ischemia, tissue ischemia, ischemia-reperfusion injury,angina pectoris, coronary artery disease, peripheral vascular disease,peripheral arterial disease, stroke, ischemic stroke, chronic wounds,diabetic wounds, myocardial infarction, congestive heart failure,pulmonary infarction, skin ulcer, etc.

Notwithstanding the above, a pathological condition in the context ofthe invention may be characterized by damage or dysfunction ofendothelial cells, i.e. wound. Non-limiting examples of wounds which maybe treated by the use of the preparation according to the presentinvention are chronic wounds, diabetic wounds, ulcer, burns,inflammatory skin disease and bowel disease.

The terms “internal condition”, “internal inflammatory condition” and“internal conditions associated with ischemia” relate to conditions anddiseases which occur inside the body of an individual that are caused byacute or latent hypoxia and inflammation in mammal end organs necessaryfor optimal functioning (e.g. bone, heart, liver, kidney, cerebrum, skinintegrity).

“Physiological solution”, as used herein, refers to a solutionexhibiting an osmotic pressure which does not lead to the destruction ofthe PBMCs or subsets thereof and can be directly administered to anindividual.

The term “free of PBMC-proliferating and PBMC-activating substances”refers to the physiological solution which does not comprise substanceswhich activate PBMCs and induce the proliferation of PBMCs or subsetsthereof. Non-limiting examples of such substances include PHA, LPS, andthe like.

According to a preferred embodiment of the present invention theinflammatory condition is selected from the group of mammal diseasesthat are related to hypoxia and inflammation of functional end organs.

According to a particularly preferred embodiment of the presentinvention the internal inflammatory condition, preferably the internalcondition associated with ischemia, is selected from the groupconsisting of myocardial ischemia, limb ischemia, tissue ischemia,ischemia-reperfusion injury, angina pectoris, coronary artery disease,peripheral vascular disease, peripheral arterial disease, stroke,ischemic stroke, myocardial infarction, congestive heart failure,trauma, bowel disease, mesenterial infarction, pulmonary infarction,bone fracture, tissue regeneration after dental grafting, auto-immunediseases, rheumatic diseases, transplantation allograft and rejection ofallograft.

The subset of peripheral blood mononuclear cells (PBMCs) is preferably Tcells, B cells or NK cells (i.e., lymphocytes). Of course it is alsopossible to use combinations of these lymphocyte cells: T cells and Bcells; T cells and NK cells; B cells and NK cells; T cells, B cells andNK cells. Methods for providing and isolating said cells are known.

It surprisingly turned out that the PBMCs of the present invention canbe cultivated in any kind of solution provided that said solution doesnot comprise substances which are not pharmaceutically acceptable, leadto an immediate death of the PBMCs, activate PBMCs and stimulate theproliferation of PBMCs (as defined above). Therefore, the solution to beused at least exhibits osmotic properties which do not lead to lysis ofthe PBMCs. The physiological solution is preferably a physiological saltsolution, preferably a physiological NaCl solution, whole blood, a bloodfraction, preferably serum, or a cell culture medium.

The cell culture medium is preferably selected from the group consistingof RPMI, DMEM, X-vivo and Ultraculture.

According to a particularly preferred embodiment of the presentinvention the cells of the present invention are cultivated under stressinducing conditions.

The term “under stress inducing conditions”, as used herein, refers tocultivation conditions leading to stressed cells. Conditions causingstress to cells include among others heat, chemicals, radiation,hypoxia, osmotic pressure (i.e. non-physiological osmotic conditions)etc.

Additional stress to the cells of the present invention leads to afurther increase of the expression and secretion of substancesbeneficial for treating internal inflammatory conditions, preferablyinternal conditions associated with ischemia.

According to a preferred embodiment of the present invention the stressinducing conditions include hypoxia, ozone, heat (e.g. more than 2° C.,preferably more than 5° C., more preferably more than 10° C., higherthan the optimal cultivation temperature of PBMCs, i.e. 37° C.),radiation (e.g. UV radiation, gamma radiation), chemicals, osmoticpressure (i.e. osmotic conditions which are elevated at least 10% incomparison to osmotic conditions regularly occurring in a body fluid, inparticular in blood), pH shift or combinations thereof.

If radiation is used to stress the PBMCs of the present invention thecells are preferably irradiated with at least 10 Gy, preferably at least20 Gy, more preferably at least 40 Gy, whereby as source Cs-137 Cesiumis preferably used.

According to a preferred embodiment of the present invention thenon-activated PBMCs or a subset thereof are cultivated in a medium forat least 4 h, preferably for at least 6 h, more preferably for at least12 h.

The pharmaceutical preparation according to the present invention can beadministered in various ways depending on the condition to be treated.Therefore, said preparation is preferably adapted for subcutaneousadministration, intramuscular administration, intra-organ administration(e.g. intramyocardial administration) and intravenous administration.

A pharmaceutical preparation according to the present invention maycomprise pharmaceutically acceptable excipients such as diluents,stabilizers, carriers etc. Depending on the administration route thepreparation according to the present invention is provided in arespective dosage form: injection solution, etc. Methods for preparingthe same are well known to the skilled artisan.

In order to increase the shelf-life of the preparation according to thepresent invention the solution a) or the supernatant b) is lyophilized.Methods for lyophilizing such preparations are well known to the personskilled in the art.

Prior its use the lyophilized preparation can be contacted with water oran aqueous solution comprising buffers, stabilizers, salts etc.

Another aspect of the present invention relates to the use of apreparation as defined above for the manufacture of a medicament fortreating an internal inflammatory condition, preferably an internalcondition associated with ischemia.

Yet another aspect of the present invention relates to a method forpreparing a pharmaceutical preparation as disclosed herein comprisingthe steps of

a) providing peripheral blood mononuclear cells (PBMCs) or a subsetthereof (e.g., lymphocytes or subset thereof),

b) culturing the cells of step a) in a physiological solution free ofPBMC-proliferating and PBMC-activating substances for at least 1 h,

c) isolating the cells of step b) and/or the supernatant thereof, and

d) preparing the pharmaceutical preparation using the cells and/or thesupernatant of step c).

The preparation according to the present invention can be obtained byincubating or culturing PBMCs in a physiological solution for at least 1h, preferably at least 4 h, more preferably at least 8 h, even morepreferably at least 12 h. In the course of this step the PBMCs begin tosynthesize and to secrete substances which are useful in the treatmentof internal inflammatory conditions. Prior, after and in the course ofthe culturing step the cells are not activated by adding PBMC activatingsubstances like PHA or LPS. After the cultivation step the cells and/orthe supernatant of the culture is isolated to be further used in thepreparation of the final pharmaceutical preparation. As discussed abovethe pharmaceutical preparation may comprise cultivated PBMCs, thesupernatant of the culture in which said cells had been incubated orboth the cultivated PBMCs as well as the culture medium.

According to a preferred embodiment of the present invention the cellsare subjected to stress inducing conditions before or in the course ofstep b), wherein said stress inducing conditions include hypoxia, ozone,heat, radiation, chemicals, osmotic pressure (e.g. induced by theaddition of salt, in particular NaCl, in order to give an osmoticpressure higher than in blood), pH shift (i.e. pH change by adding acidsor hydroxides to give a pH value of 6.5 to 7.2 or 7.5 to 8.0) orcombinations thereof.

According to a preferred embodiment of the present invention the cellsare irradiated before or in the course of step b) with at least 10 Gy,preferably at least 20 Gy, more preferably at least 40 Gy, with ozone,with elevated temperature or with UV radiation.

Another aspect of the present invention relates to a preparationobtainable by a method as described above.

Another aspect of the present invention relates to a method for treatinginternal inflammatory conditions, preferably internal conditionsassociated with ischemia, by administering to an individual in needthereof an appropriate amount of the pharmaceutical preparationaccording to the present invention. Depending on the condition to betreated the preparation of the present invention is administeredintramuscularly, intravenously, intra-organly (e.g. intramyocardially)or subcutaneously.

In a preferred embodiment of the present invention the pharmaceuticalpreparation comprises at least 500, preferably at least 1000, morepreferably at least 10⁵, even more preferably at least 10⁶ PBMCs per mLobtainable by a method as outlined above. Correspondingly at least 500,preferably at least 1000, more preferably at least 10⁵, even morepreferably at least 10⁶, PBMCs are administered to an individual to betreated.

The present invention is further illustrated by the following figuresand examples, however, without being restricted thereto.

FIGS. 1A and 1B show: FIG. 1A: the study protocol and the time points ofevaluation of cardiac function by echocardiography, histology andimmunohistology; FIG. 1B: the percentage of irradiated andnon-irradiated rat PBMCs positively stained for Annexin after a cultureperiod of 18 h.

FIG. 2A: FACS analysis shows that irradiation leads to induction ofapoptosis in human PBMCs with a time dependent increase of Annexinexpression over 48 h. FIG. 2B: Co-incubation of LPS stimulated PBMCs ormonocytes with irradiated apoptotic autologous PBMCs demonstrates areduced secretion of the proinflammatory cytokine IL-1β in a dosedependent manner. FIG. 2C: To a lesser extent this finding alsocorrelates with the IL-6 secretion profile of LPS stimulated PBMCs andmonocytes in the presence of IA-PBMC. FIG. 2D: Addition of autologousIA-PBMCs in a mixed lymphocyte reaction with LPS stimulation decreasesT-cell proliferation as measured by counts per minute (cpm). FIG. 2E:RT-PCR RNA expression analysis of VEGF, IL-8/CXCL8, and MMP transcriptsshows an upregulation of IL-8/CXCL8 and especially MMP9 in irradiatedPBMCs after a culture period of 24 h. FIG. 2F: ELISA analysis of VEGF,IL-8/CXCL8, and MMP9 demonstrates that MMP9 is predominantly found incell lysates whereas differences in VEGF and IL-8/CXCL8 proteinsecretion remain approximately at the same level in both viable cellsand IA-PBMC. FIG. 2G: Human fibroblasts incubated in supernatantsobtained from cell cultures of viable or IA-PBMCs exhibit a strongupregulation of VEGF, IL-8/CXCL8, and MMP9 transcripts in RT-PCRanalysis, peak values were found in fibroblasts incubated in IA-PBMCsupernatants.

FIGS. 3A-3C: CFSE labeled syngeneic PBMCs administered via the tail veinin rats after artificial myocardial infarction were predominantly foundin the spleen (FIG. 3B), to a lesser extent in the liver (FIG. 3A), andno cells in the infarcted heart (FIG. 3C). FIGS. 3D-3F: HE stainedinfarct zones of rats injected with either medium (FIG. 3D) or viablePBMCs (FIG. 3E) show a comparable pattern of ischemic myocardiuminfiltrated by immune cells, tissues obtained from rats receivingIA-PBMCs indicate very dense infiltrations. FIGS. 3G-3I: Rats treatedwith viable cells (FIG. 3H) reveal slightly more of CD68+ stained cellsin the infarcted than in medium treated rats (FIG. 3G), but a 3-foldhigher amount of CD68+ was detected in IA-PBMC injected animals. FIG.3J-3L: Higher levels of S1000+ cells were found in rats receiving mediumalone compared to the application of viable PBMCs or IA-PBMCs.

FIGS. 4A-4C: Almost 4-fold higher amounts of cells staining positive forVEGF were detected in infarcted myocardial tissue obtained from animalsinjected with IA-PBMCs (FIG. 4C), in comparison with medium (FIG. 4A) orviable cell treatment (FIG. 4B). FIGS. 4D-4F: A similar expressionpattern was found for VEGF receptor KDR/FLK1 with peak values in theIA-PBMC group (FIG. 4F) compared to medium (FIG. 4D) and viable cells(FIG. 4E). FIGS. 4G-4I: No differences were detected for CD34 in allthree groups. FIGS. 4J-4L: immunohistological analysis for the markerc-kit in infarcted hearts shows a high quantity of positively stainedcells and dense localization in rats injected with IA-PBMCs (FIG. 4L)and fewer cells in medium (FIG. 4J) and viable cell receiving animals(FIG. 4K).

FIGS. 5A-5C: Histological analysis of ischemic rat hearts explanted 6weeks after induction of myocardial infarction (Elastica van Giesonstaining), hearts from medium injected animals (FIG. 5A) appear moredilated and show a greater extension of fibrotic tissue, scar extensionwas reduced in viable cell injected rats (FIG. 5B) with fewer signs ofdilatations, the least amount of scar tissue formation was detected inIA-PBMC injected animals (FIG. 5C). FIG. 5D: statistical analysis ofdata obtained from planimetric analysis of specimen collected 6 weeksafter LAD-ligation shows a mean scar extension of 24.95%±3.6 in medium,of 14.3%±1.3 in viable PBMC and 5.8%±2 in IA-PBMC injected animals(mean+SEM). FIG. 5E-5G: Assessment of cardiac function parametersshortening fraction, ejection fraction and endsystolic diameter byechocardiography evidences a better recovery after myocardial infarctionin animals injected with IA-PBMC.

FIG. 6A shows that neither unstimulated viable PBMCs or IA-PBMCs secretethe mainly monocyte derived pro-inflammatory cytokine TNF-α.(Significances are indicated as follows: *p=0.05, **p=0.001; n=8)

FIG. 6B demonstrates a strong induction of pro-inflammatory Interferon-γsecretion after activation as compared to unstimulated PBMC.(Significances are indicated as follows: *p=0.05, **p=0.001; n=8)

FIG. 7A shows pooled results of flow cytometric analysis. PBMCs weregated for T cells and expression of activation markers CD69 and CD25were evaluated. (Significances are indicated as follows: *p=0.05,p=0.001; n=4)

FIG. 7B displays a representative FACS analysis of PBMCs eitheractivated (PHA, CD3 mAb). Gating represents % of positive cells.

FIG. 8 shows high proliferation rates as measured by 3[H]-thymidineincorporation of stimulated PBMCs when compared to viable PBMCs culturedin RPMI without stimulation.

FIG. 9 shows inhibition of T cell response of PBMC secretoma in T cellproliferation assays.

FIGS. 10A-10F show supernatant levels of Interleukin-8, Gro-alpha,ENA-78, ICAM-1, VEGF, and Interleukin-16. Apoptotic PBMCs show amarkedly different secretion pattern of these cytokines and chemokinerelated to angiogenesis and immune suppression compared to viable cells.This effect was even more pronounced when cells were incubated at highdensities.

FIG. 11 shows the extension of myocardial scar tissue 6 weeks afterexperimental LAD ligation (as % of the left ventricle). Animals thatwere infused with cell culture supernatants derived from apoptotic cellsevidence a significant reduction of collagen deposition, less scarextension and more viable myocardium.

FIGS. 12A-12C show macroscopic appearance of rat hearts explanted 6weeks after experimental myocardial infarction. Animals transfused withsupernatants from irradiated apoptotic cells (FIG. 12C) evidencedreduced collagen deposition and much smaller infarcted areas compared tomedium (FIG. 12A) or supernatants from viable cells (FIG. 12B). Scartissue is colored in green for better visualization.

FIGS. 13A-13D show representative echocardiographic analyses (M-Mode).Cardiac function was significantly better in rats transfused withIA-PBMC supernatants (FIG. 13C) compared to medium (FIG. 13A) and viablecell treated rats (FIG. 13B). Echocardiographic imaging from a shamoperated rat is depicted in FIG. 13D.

FIGS. 14A and 14B show echocardiographic analyses conducted 6 weeksafter myocardial infarction. Rats therapied with supernatants fromirradiated apoptotic PBMCs evidence a significantly better cardiacfunction compared to medium or viable cell culture supernatant infusedanimals.

FIG. 15 shows Kaplan-Meier survival curve for all four treatment groups.Both viable or apoptotic PBMC cell culture supernatant infused animalsevidence a better survival compared to medium injected rats. (p<0.1).

FIG. 16 shows anti-CD3 and PHA stimulation experiments performed withPBMC.

FIG. 17A-17C shows the proliferation of PBMCs upon stimulation withanti-CD3, PHA and mixed lymphocytes.

FIGS. 18A and 18B show the level of Annexin V and PI positivity of thesupernatant of CD4+ cells incubated with PBMC supernatants.

FIGS. 19A and 19B show the inhibition of the up-regulation of CD25 andCD69 in CD4+ cells by PBMC supernatant.

FIG. 20 shows that the demonetizing of IL-10 and TGF-β did not increasethe proliferation rates of CD4+ cells.

EXAMPLES Example 1

Acute myocardial infarction (AMI) often leads to congestive heartfailure. Despite current pharmacological and mechanicalrevascularization no effective therapy is defined experimentally toreplace infarcted myocardium. Integral components of the remodelingprocess after AMI are the inflammatory response and the development ofneo-angiogenesis after AMI. These processes are mediated by cytokinesand inflammatory cells in the infarcted myocardium that phagocytoseapoptotic and necrotic tissue and initiate homing of interstitialdendritic cells (IDC) and macrophages. Clinical trials aimed toattenuate AMI induced inflammatory response were abducted since systemicimmune suppression (steroids) led to increased infarct size and delayedmyocardial healing. From these data it was concluded that inflammatoryresponse after AMI is responsible for tissue stabilization and scarformation. A new field in regenerative cardiovascular medicine emergedwhen investigators observed that distant stem cells sense sites ofdamage and promote structural and functional repair. By utilizing thisapproach, Orlic et al. injected c-kit positive endothelial progenitorcells (EPC) into the boarder zone of experimental AMI and increasedneo-angiogenesis and regeneration of myocardial and vascular structures.This work ignited a plethora of publications that demonstrated aregenerative potential of “cell based therapy”, however it still remainselusive whether this therapeutic effect is caused by the transplantedcells themselves, recruitment of resident cardiac stem cells, or byactivation of, as yet, unidentified paracrine and immunologicmechanisms. Ischemia in infarcted myocardium causes apoptotic processesand initiates alterations of cell surface lipids on dying cells. Thebest-characterized modification is the loss of phospholipid asymmetryand exposure of phosphatidylserine (PS). These PS are recognized bymacrophages and dendritic cells (antigen presenting cells, APC) vialigands such as thrombospondin, CD14 and CD36. Under physiologicalconditions these receptors serve to engulf apoptotic and necrotic debrisand initiate a silent “clean up” process. This process of phagocytosisby APC leads to a phenotypic anti-inflammatory response as determined byaugmented IL-10 and TGF-β production and impaired APC function. Ofclinical relevance are reports that demonstrated that infusion ofapoptotic cells lead in a hematopoietic cell (HC) transplantation modelto allogeneic HC engraftment and to a delay of lethal acutegraft-versus-host disease (GVHD). Moreover, in solid organtransplantation models infusion of donor apoptotic cells increased heartgraft survival. Contrary to inflammation and relevant to progenitor cellrecruitment from bone marrow (BM) it was shown that opsonization ofapoptotic cells elicits enhanced VEGF and CXC8/IL-8 production of APC.In addition to the latter cytokines MMP9 was also identified to be vitalfor EPC recruitment and liberation from the bone marrow.

The current “status quo” in AMI treatment is directed toward earlyreperfusion and reopening the acute occluded coronary artery and thatmyocardial inflammation post infarction is perceived beneficiary despitethis condition increases myocardial damage and counteracts endogenousrepair mechanisms.

Material and Methods Induction of Apoptosis of PBMCs and Generation ofSupernatants

For the in vivo experiments blood was drawn from healthy youngvolunteers. Apoptosis was induced by Cs-137 Cesium irradiation with 60Gy (human PBMC) or with 45 Gy for in vivo (rat PBMC) experiments. Cellswere resuspended in serum free Ultra Culture Medium (Cambrex Corp., USA)containing 0.2% gentamycin-sulfate (Sigma Chemical Co, USA), 0.5%β-mercapto-ethanol (Sigma, USA), 1% L-glutamine (Sigma, USA) andcultured in a humidified atmosphere for 24 h for in vitro experiments(concentration of cells, 1×10⁶ mL). Induction of apoptosis was measuredby Annexin V-fluorescein/propidium iodide (FITC/PI) co-staining (BectonDickinson, USA) on a flow cytometer. Annexin-positivity of PBMCs wasdetermined to be >70% and are consequently termed IA-PBMC.Non-irradiated PBMCs served as controls and are termed viable-PBMC. Fromboth experimental settings supernatants were collected and served asexperimental entities as described below (SN-viable-PBMC, SN-IA-PBMC).

LPS-Stimulation Experiments

Human PBMCs and monocytes (purity >95%) were separated using a magneticbead system (negative selection Miltenyi Biotec, USA). PBMCs andmonocytes were co-incubated for 4 h with different concentrations ofapoptotic autologous PBMCs (annexin positivity >70%) andLipopolysaccharide (1 ng/mL LPS; Sigma Chemical Co, USA). Supernatantswere secured and kept frozen at −80° C. until further tests. IL-6 andIL-1β release was determined using commercially available ELISA kits(BenderMedSystems, Austria).

Monocyte-Derived DC Preparation and T-Cell Stimulation

PBMCs were isolated from heparinized whole blood of healthy donors bystandard density gradient centrifugation with Ficoll-Paque (GEHealthcare Bio-Sciences AB, Sweden). T cells and monocytes wereseparated by magnetic sorting using the MACS technique (MiltenyiBiotec). Purified T cells were obtained through negative depletion ofCD11b, CD14, CD16, CD19, CD33, and MEC class II-positive cells with therespective monoclonal antibody. Monocytes were enriched by using thebiotinylated CD14 mAb VIM13 (purity 95%). DCs were generated byculturing purified blood monocytes for 7 days with a combination ofGM-CSF (50 ng/mL) and IL-4 (100 UmL). Subsequently, DCs were differentlystimulated. Maturation was induced either by adding 100 ng/mL LPS fromEscherichia coli (serotype 0127-B8, Sigma Chemie) for 24 h alone or byadding LPS for 2 h and further culturing the dendritic cells withapoptotic cells in a 1:1 ratio for 22 h. Additionally, DCs were treatedwith apoptotic cells alone (1:1) for 24 h. For the mixed leukocytereaction (MLR), allogenic, purified T cells (1×10⁵/well) were incubatedin 96-well cell culture plates (Corning Costar) with graded numbers ofdifferently stimulated DCs for 6 days. The assay was performed intriplicate. Proliferation of T cells was monitored by measuring[methyl-3H]thymidine (ICN Pharmaceuticals) incorporation, added after 5days. Cells were harvested after 18 h and incorporated[methyl-3H]thymidine was detected on a microplate scintillation counter.

Cell Culture, RNA Isolation and cDNA Preparation of Viable PBMCs,IA-PBMCs, and SN Exposed Fibroblasts

IA-PBMCs, viable-PBMCs (1×10⁶ cells, both conditions cultured for 24 hin Ultra Culture Medium), and fibroblasts exposed toSN-viable-PBMC/SN-IA-PBMC were investigated (1×10⁵ fibroblasts obtainedfrom Cascade Inc. (USA) were cultured in Dulbecco's modified Eaglemedium (DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, PAA, Austria), 25 mM L-glutamine (Gibco, BRL, USA) and 1%penicillin/streptomycin (Gibco) and seeded in 12 well plates;fibroblasts were co-incubated with SN-viable-PBMC, SN-IA-PBMC for 4 and24 h respectively). After RNA extraction of PBMCs and fibroblasts (usingRNeasy, QIAGEN, Austria) following the manufacturer's instruction, cDNAswere transcribed using the iScript cDNA synthesis kit (BioRad, USA) asindicated in the instruction manual.

Quantitative Real Time PCR

mRNA expression was quantified by real time PCR with LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science, Penzberg, Germany)according to the manufacturer's protocol. The primers for VEGF were:forward: 5′-CCCTGATGAGATCGAGTACATCTT-3′ (SEQ ID NO: 1), reverse:5′-ACCGCCTCGGCTTGTCAC-3′ (SEQ ID NO: 2); for IL-8 forward:5′-CTCTTGGCAGCCTTCCTGATT-3′ (SEQ ID NO: 3), reverse:5′-TATGCACTGACATCTAAGTTCTTTAGCA-3′ (SEQ ID NO: 4); for MMP9 forward:5′-GGGAAGATGCTGGTGTTCA-3′ (SEQ ID NO: 5), reverse:5′-CCTGGCAGAAATAGGCTTC-3′ (SEQ ID NO: 6) and for β-2-microglobulin β 2M,forward: 5′-GATGAGTATGCCTGCCGTGTG-3′ (SEQ ID NO: 7), reverse:5′-CAATCCAAATGCGGCATCT-3′ (SEQ ID NO: 8). The relative expression of thetarget genes was calculated by comparison to the house keeping gene β2Musing a formula described by Wellmann et al. (Clinical Chemistry. 47(2001) 654-660, 25). The efficiencies of the primer pairs weredetermined as described (A. Kadl, et al. Vascular Pharmacology. 38(2002) 219-227).

Release of Pro-Angiogenetic Factors and MMP9 by Viable PBMCs andIA-PBMCs after Culture

IA-PBMCs (5×10⁵) and viable PBMCs were incubated in a humidifiedatmosphere for 24 h. Supernatants were collected after 24 h andimmediately frozen at −80° C. until evaluation. Lysates of respectivecells served as controls. Release of pro-angiogenetic factors (VEGF-A,CXCL-8/IL-8, GMCSF, GCSF) and MMP9, an accepted liberating factor ofc-kit cells, were analyzed utilizing ELISA (R&D, USA) following themanufacturer's instructions. Plates were read at 450 nm on a WallacMultilabel counter 1420 (PerkinElmer, USA).

Acquisition of Syngeneic IA-PBMCs and Viable-PBMCs for AMI In VivoExperiment

Syngeneic rat PBMCs for in vivo experiments were separated by densitygradient centrifugation from whole-blood obtained from prior heparinizedrats by punctuation of the heart. Apoptosis was induced by Cs-137 Cesiumirradiation with 45 Gy for in vivo experiments and cultured for 18 h asdescribed above. (annexin staining >80% IA-PBMC, annexin staining <30%viable PBMC, 1×10⁶/mL).

Induction of Myocardial Infarction

Myocardial infarction was induced in adult male Sprague-Dawley rats byligating the LAD as previously described (Trescher K, et al. CardiovascRes. 2006: 69(3): 746-54). In short, animals were anesthetizedintraperitoneally with a mixture of xylazin (1 mg/100 g bodyweight) andketamin (10 mg/100 g bodyweight) and ventilated mechanically. A leftlateral thoracotomy was performed and a ligature using a 6-0 prolene wasplaced around the LAD beneath the left atrium. Immediately after theonset of ischemia 8×10⁶ apoptotic PBMCs suspended in 0.3 mL cell culturemedium were infused through the tail vein. Infusion of cell culturemedium alone, viable PBMCs, and sham operation respectively served inthis experimental setting as negative controls. The rat experimentaldesign is shown in FIG. 1 (FIGS. 1A and 1B).

Tracking of Apoptotic Cells

8×10⁶ syngeneic rat PBMCs were labelled with 15 μM Carboxy-fluoresceindiacetate succinimidyl ester (CFSE, Fluka Bio-Chemika, Buchs,Switzerland) at room temperature for 10 min. Labelling was stopped bythe addition of fetal calf serum (FCS). Apoptosis was induced (annexinV>70%) and cells were injected after ligation procedure. 72 h afteroperation rats were sacrificed and liver, spleen and heart wereprocessed following a standard procedure for frozen sections (n=4).Samples were analyzed by confocal laser scanning microscopy (ZEISS LSM510 laser scanning microscope, Germany) as described previously(Kerjaschki D, J Am Soc Nephrol. 2004; 15: 603-12).

Histology and Immunohistochemistry In Vivo

All animals were sacrificed either 72 h or 6 weeks after experimentalinfarction. Hearts were explanted and then sliced at the level of thelargest extension of infarcted area (n=8-10). Slices were fixed with 10%neutral buffered formalin and embedded in paraffin for (immune-)histological staining. The tissue samples were stained withhematoxylin-eosin (H&E) and elastic van Gieson (evg). Immunohistologicalevaluation was performed using the following antibodies directed to CD68(MCA 341R, AbD Serotec, UK), VEGF (05-443, Upstate/Milipore, USA), Flk-1(sc-6251, Santa Cruz Biotechnology, USA), CD34 (sc-52478, Santa CruzBiotechnology, USA), c-kit (sc-168, Santa Cruz Biotechnology, USA), 5100beta (sc-58841, Santa Cruz Biotechnology, USA). Tissue samples wereevaluated on an Olympus Vanox AHBT3 microscope (Olympus Vanox AHBT3,Olympus Optical Co. Ltd., Japan) at 200× magnification and captureddigitally by using a ProgRes Capture-Pro C12 plus camera (Jenoptik LaserOptik Systeme GmbH, Germany).

Determination of Myocardial Infarction Size by Planimetry

In order to determine the size of the infarcted area, Image J planimetrysoftware (Rasband, W. S., Image J, U. S. National Institutes of Health,USA) was used. The extent of infarcted myocardial tissue (% of leftventricle) was calculated by dividing the area of the circumference ofthe infarcted area by the total endocardial and epicardialcircumferenced areas of the left ventricle. Planimetric evaluation wascarried out on tissue samples stained with evg for better comparison ofnecrotic areas. Infarct size was expressed as percent of total leftventricular area.

Cardiac Function Assessment by Echocardiography

Six weeks after induction of myocardial infarction rats wereanaesthetized with 100 mg/kg Ketamin and 20 mg/kg Xylazin. Thesonographic examination was conducted on a Vivid 5 system (GeneralElectric Medical Systems, USA). Analyses were performed by anexperienced observer blinded to treatment groups to which the animalswere allocated (EW). M-mode tracings were recorded from a parasternalshort-axis view and functional systolic and diastolic parameters wereobtained. Ventricular diameters and volumes were evaluated in systoleand diastole. Fractional shortening was calculated as follow: FS(%)=((LVEDD-LVESD)/LVEDD)×100%

Statistical Methods

Statistical analysis was performed using SPSS software (SPSS Inc., USA).All data are given as mean±standard of the mean. Normal distribution wasverified using the Kolmogorov-Smirnov test. Paired two-sided t-tests fordependent, unpaired t-tests for independent variables were utilizedcalculating significances. Bonferroni-Holm correction was used to adjustp-values for multiple testing. P-values <0.05 were consideredstatistically significant.

Results

Induction of Apoptosis with Cesium Irradiation (IA-PBMC)

In order to evaluate the immunomodulatory potential of apoptotic cells,first the cellular response to induction of apoptosis by cesiumirradiation of human peripheral blood mononuclear cells (PBMC) wasdetermined by flow cytometry utilizing Annexin-V/PI staining on a flowcytometer. Irradiation caused positivity for Annexin on PBMCs in a timedependent manner and peaked within 24 h as compared to viable PBMC.Viable cells served as controls (FIG. 2A). Since Annexin-V binding washighest after 24 h all further in vitro investigations were performedafter this culture period (IA-PBMC). Viable PBMCs served in RT-PCR andsupernatant experiments as control.

IA-PBMCs Evidence Immune Suppressive Features In Vitro

Interleukin-1β and IL-6 is recognized as the predominantpro-inflammatory mediator in myocardial infarction in vivo. To test thehypothesis whether IA-PBMC has an effect on cellular response humanmonocytes, PBMCs were co-incubated with IA-PBMC and target cells werestimulated with LPS. A dose dependent decrease in secretion of IL-1β andIL-6 in cultures of both cell types as evaluated by ELISA was found(FIGS. 2B and 2C). To verify anti-proliferative effects of IA-PBMC in anallogeneic model a mixed lymphocyte reaction (MLR) was utilized.Allogenic, purified T-cells were utilized and these effector cells wereincubated with graded doses of dendritic cells with/without addition ofIA-PBMC. FIG. 2D evidences that co-incubation of IA-PBMC decreasesproliferation rate in a dose-dependent manner.

IA-PBMC and Viable PBMC Evidence Increased mRNA Transcription of VEGF,IL-8/CXL8, and MMP9

To substantiate whether irradiation leads to enhanced mRNA transcriptionof proteins known to be related to mobilization of EPC PBMC was analyzedafter separation, and after apoptosis induction (24 h). Viable PBMCserved as control (viable PBMC or IA-PBMC). RNA transcription showedlittle difference VEGF expression as determined by RT-PCR, however astrong enhancement of IL-8/CXCL8 and MMP9. Peak induction for IL-8/CXL8in IA-PBMC was 6 fold versus 2 fold in viable cells, and 30 fold versus5 fold for MMP9, respectively (FIG. 2E).

IA-PBMC and Viable PBMC Secrete Paracrine Factors that Cause EndothelialProgenitor Cells (EPC) Liberation

SN derived from IA-PBMC and viable PBMC were quantified for VEGF,IL-8/CXCL8, GMCSF, GCSF and MMP9 utilizing ELISA after 24 h culture. Asseen in FIG. 2F VEGF, IL-8/CXCL8 and MMP9 evidenced an increment. GM-CSFand G-CSF were not detectable. Of interest was the finding that MMP9evidenced peak values in cell lysates.

SN Derived from IA-PBMC and Viable PBMC Augment Pro-Angiogenic mRNATranscription in Mesenchymal Fibroblasts

Since stromal cells in bone marrow are constitutively fibroblasts it wassought to investigate whether co-incubation of fibroblasts with SNderived from IA-PBMC and viable PBMC had the ability to increase VEGF,IL-8/CXCL8 and MMP9 mRNA transcription, factors responsible for EPCmobilization. RT-PCR was conducted at 4 and 24 h. Highest levels ofinduction were detected for IL-8/CXCL8 in cells cultivated in IA-PBMCSN, reaching an almost 120-fold induction at 4 h as compared to control.This response is also present at 24 h. A comparable response was foundfor VEGF, whereas MMP9 upregulation was predominantly found after 24 h.This data indicates that SN contains paracrine factors that enhancefibroblasts to augment mRNA products responsible for pro-angiogeniceffects in the BM (FIG. 2G).

Adoptive Transfer of CFSE Labelled IA-PBMC in a Rat MyocardialInfarction Model

Because it could be proven that cultured IA-PBMC are bothanti-inflammatory and pro-angiogenic in vitro IA-PBMC and viable PBMCwere infused in an acute rat AMI model. First it was sought to determinewhere these cultured cells are homing after infarction. CFSE labelledIA-PBMC were injected into the rat's tail vein shortly after LAD arteryligation. A representative histology is seen in FIGS. 3A-3C. Themajority of CFSE IA-PBMC were trapped in the spleen and liver tissuewithin 72 h. No cells were observed in the heart.

Diverted Early Inflammatory Immune Response in IA-PBMC Treated AMI

Upon closer investigation in H.E. staining, control infarction andviable leucocytes (viable PBMC) treated AMI rats evidenced a mixedcellular infiltrate in the wound areas in accordance to granulationtissue with abundance of neutrophils, macrophages/monocytes,lymphomononuclear cells, fibroblasts and activated proliferatingendothelial cells admixed to dystrophic cardiomyocytes (FIGS. 3D and 3E)within 72 h after AMI. In contrast, AMI rats treated with IA-PBMCevidenced a dense monomorphic infiltrate in wound areas that consistedof medium sized monocytoid cells with eosinophilic cytoplasm, densenuclei and a round to spindle shaped morphology (FIG. 3F). In addition,few lymphomononuclear cells, especially plasma cells, fibroblasts andendothelial could be detected. Immunohistochemical analysis revealedthat the cellular infiltrate in IA-PBMC AMI rats was composed ofabundant CD68+ monocytes/macrophages (FIG. 3I) that were much weaker inthe other two groups (MCI, Viable PBMC, IA-PBMC, high power field, HPF,60.0±3.6, 78.3±3.8, 285.0±23.0 (SEM), respectively) (FIGS. 3G and 3H).Content on Vimentin positive mesenchymal cells was similar in all groupswhile S100+ dendritic cells were preferentially found in controlinfarction (AMI, Viable PBMC, IA-PBMC, HPF 15.6±1.7, 12.4±2.3, 8.4±1.2(SEM), respectively as compared to treated groups (FIGS. 3J, 3K, 3HRepresentative histology, n=5)).

Early Homing of VEGF+, Flk1+ and c-Kit+ Cells in IA-PBMC Treated AMI

Since IA-PBMC evidenced a dense monomorphic infiltrate in wound areasthat consisted of medium sized monocytoid cells with eosinophiliccytoplasm and dense nuclei multiple surface markers related toneo-angiogenesis and regenerative potency were explored. This cellpopulation identified in the H.E. staining in IA-PBMC treated AMI groupstained highly positive for vascular endothelial growth factor (VEGFa),Flk-1 and c-kit (CD 117) (FIG. 4C, 4F, 4I). Expression of both markerswas reduced in control AMI and viable PBMC treated AMI group (FIGS. 4A,4B, 4D 4E, 4J). Interestingly, IA-PBMC treated AMI evidenced increasedCD34+ cells within the densely populated infarcted area which isattributed to vascular structure putatively referring to colonization of34+ cells (I) as compared to control (FIGS. 4G, 4H) (Representativehistology, n=5)

Attenuated Infarct Size in IA-PBMC Treated AMI

In a planimetric analysis performed on EVG stained tissue samples fromhearts explanted 6 weeks after myocardial infarction was induced, ratsreceiving saline show a collagenous scar extending to over 24.95%±3.58(SEM) of the left ventricle with signs of dilatation. In IA-PBMC treatedrats these signs were almost abrogated with infarct sizes of 5.81%±2.02(SEM) as compared to 14.3%±1.7 (SEM) treated with viable PBMC (FIGS.5A-5C).

LV Function Improves in IA-PBMC Treated AMI

Intravenous application of syngeneic cultured IA-PBMC significantlyimproves echocardiographic parameters as compared with viable PBMC orculture medium treated animals. Shortening fraction (SF) evidencedvalues of 29.16%±4.65 (SEM) in sham operated animals, 18.76%±1.13 (SEM)in medium treated AMI animals, 18.46%±1.67 (SEM) in the viable PBMC AMIgroup and 25.14%±2.66 (SEM) in IA-PBMC treated rats (FIG. 5E). Ejectionfraction (EF) was 60.58%±6.81 (SEM) in sham operated rats and declinedto 42.91% 12.14 (SEM) in AMI animals treated with medium, and to42.24%±3.28 (SEM) in animals receiving viable PBMC, whereas rats treatedwith IA-PBMCs evidenced an EF of 53.46%±4.25.

Analysis of end-systolic and end-diastolic diameters (LVESD, LVEDD),end-systolic and end-diastolic volumes (LVESV, LVEDV) showed acomparable pattern to the previously observed values. Saline receivinganimals and viable PBMC treated rats showed LVEDD values of 10.43mm±0.21 (SEM) and 11.03 mm±0.40 respectively, IA-PBMC rats evenrepresented a slightly reduced left-ventricular diastolic diameter of8.99 mm±0.32 compared to 9.47 mm 0.64 in sham operated animals.Differences in systolic diameters were less pronounced, but in the sameranking (Panel 5 (a, b, c)

Conclusion:

These findings demonstrate that irradiated apoptotic PBMCs (IA-PBMCs)induce immune suppression in vitro and is associated to secretion ofpro-angiogenic proteins. Therefore cultured viable-PBMCs and IA-PBMCswere infused in an acute rat AMI model and demonstrated that thistreatment evoked massive homing of FLK1+/c-kit+ positive EPC intoinfarcted myocardium within 72 h and caused a significant functionalrecovery within 6 weeks.

Co-culture of IA-PBMCs in immune assays resulted in reduced IL-1β andIL-6 production and attenuated allogeneic dendritic mixed lymphocytereaction (MLR). Both immune parameters were described to have a role ininflammation after myocardial ischemia. In addition, it was evidencedthat viable- and IA-PBMCs secrete CXCL8/IL-8 and MMP9 into the culturemedium within 24 h. These proteins were described to be responsible forneo-angiogenesis and recruitment of EPC from the BM to the ischemicmyocardium. The CXCL8/IL-8 chemokine belongs to the CXCL family thatconsists of small (<10 kDa) heparin-binding polypeptides that bind toand have potent chemotactic activity for endothelial cells. Three aminoacid residues at the N-terminus (Glu-Leu-Arg, the ELR motif) determinebinding of CXC chemokines such as IL-8 and Gro-alpha to CXC receptors 1and 2 on endothelial cells and are promoting endothelial chemotaxis andangiogenesis. In addition MMP9 secretion was identified to be pivotal inEPC mobilization since this matrixproteinase serves as signal to releasesoluble kit-ligand (sKitL), a chemokine that causes the transition ofendothelial and hematopoietic stem cells (EPC) from the quiescent toproliferative niche in the BM. In a further in vitro assay it could bedemonstrated that the supernatant (SN) derived from cultured viable- andIA-PBMCs had the ability to enhance mRNA transcription of CXCL8/IL-8 andMMP9 in mesenchymal fibroblasts. These data indicate that SN derivedfrom viable and irradiated PBMCs contain paracrine factors that confer abiological situation in the BM which results in elution of c-kit+ EPCinto circulation.

In order to prove any beneficial effect of this culture-cell suspensionin vivo a model of open chest myocardial injury and infused culturedviable- and IA-PBMCs shortly after LAD ligation in rat animal model wasutilized. In a first attempt it was proved that CSFE labelled IA-PBMCswere trapped in majority in the spleen and the liver. These dataindicate that “cell-based therapy” does not home in infarctedmyocardium. In contrary, it is much more likely that paracrine effects,either by “modified” culture medium alone or by evoked “immune mediatedcytokine storm” due to cell-culture suspension exposure is causative forthe regenerative effect in AMI. Since immediate inflammation after acuteischemia determines the road map to ventricular dilatation histologicalanalysis after 72 h after AMI was performed. It could be shown thatIA-PBMCs treated rats evidenced massive homing of CD68+ andVEGFa/FLK1/c-kit+ positive EPC cell populations within this time period.In contrast, more S100 β positive dendritic cells were found in controlAMI, indicating enhanced APC based inflammation in control AMI.

The results seen in IA-PBMCs treated rats partly foil currently acceptedknowledge about the natural course of myocardial infarction. In regardsto inflammation: Under normal conditions remodeling processes aremediated by cytokines and inflammatory cells in the infarcted myocardiumthat initiate a wound reparation process that is landmarked byphagocytosis and resorption of the necrotic tissue, hypertrophy ofsurviving myocytes, angiogenesis and, to a limited extent, progenitorcell proliferation. Any experimental approach so far that intervenedinto inflammatory response post infarction was shown to be detrimentalin AMI models. When interpreting the present histological short termdata it is argued that IA-PBMC cell-medium suspension in AMI results inan advanced transitioning from inflammation to c-kit+ EPC repair phase.Previous work has confirmed that bone marrow of circulating progenitorcell therapy after AMI improve cardiac function, regardless of whethertransdifferentiation of the cells to cardiomyocytes occurs or does not.In regard to c-kit+ EPC the bone marrow derived cells are considered ashaving a significant role as indispensable for cardiac repair.Pharmacological inhibition with imatinib mesylate and non-mobilizationof c-kit+ EPC resulted in an attenuated myofibroblast response after AMIwith precipitous decline in cardiac function.

These results show the regenerative potency of infusing “syngeneic”cultured IA-PBMCs in patients suffering from AMI and indicate thatpatients who suffer from acute AMI would benefit from being transfusedwith autologous (=from the patient to be treated or from the samespecies) IA-PBMC.

Example 2 Resting Peripheral Blood Mononuclear Cells (PBMC) Evidence LowActivation Marker and Reduced Inflammatory Cytokine Production

Activated peripheral blood mononuclear cells (PBMCs) and theirsupernatants (SN) are supposed to be beneficial in wound regeneration(Holzinger C et al. Eur J Vasc Surg. 1994 May; 8(3): 351-6.). In example1 it could be shown that non-activated PBMCs and SN derived thereof hasbeneficial effects in an experimental acute myocardial infarct (AMI) andwounding model. Since non-activation of PBMCs has to be verifiedexperimentally it was investigated whether cultivation of PBMCs led toenhanced T-cell activation markers (CD69, CD25) or enhanced inflammatorycytokine secretion (monocyte activation=TNFα, T-cell activation=INF-γ).In a control experiment cultured T cells were triggered by CD3 mAbstimulation or Phytohemagglutinin (PHA).

Methods and Results

Venous blood was collected in EDTA-tubes from healthy volunteers. AfterFicoll-Hypaque density grade separation, PBMCs were collected anddivided into viable and irradiated apoptotic cells (IA-PBMC). To obtainapoptotic cells, PBMCs were irradiated with 60 Gy (Caesium-137). Forflow cytometric analysis 500,000 PBMCs were cultivated in 200 μlserum-free medium. Cells were either stimulated with PHA (7 μg/mL) orCD3-mAb (10 μg/mL) or were left unstimulated. After 24 h of incubationcells were washed, stained for CD3, CD69 and CD25 (R&D System) andevaluated for surface activation markers on a FC500 (Coulter). For ELISAassays, PBMCs were cultivated overnight at a density of 2.5×10⁶cells/mL, either with or without PHA or CD3 stimulation. After 24 hsupernatants were harvested and frozen at −20° C. Commercially availableELISA kits for TNF-α (R&D) and INF-γ (Bender) were purchased. In short,MaxiSorp plates were coated with antibodies against INF-α and INF-γ andstored overnight. After 24 h, plates were washed and samples added induplicates to each well. After incubation and addition of a detectionantibody and Streptavidin-HRP, TMB-substrate was added to each well.After color development, the enzymatic reaction was stopped by additionof sulfuric acid. Optical density values were read on a Wallac Victor3plate reader.

Results:

FACS analysis: CD3 and PHA stimulated T cells showed an upregulation ofactivation markers CD69 and CD25 after 24 h of incubation. Unstimulatedand apoptotic cells expressed only low amounts of CD69 and CD25 (FIG. 6A(representative sample, FIG. 6B, histogram, n=4). Statisticalsignificance is indicated by asterisks (**p<0.001, *p<0.05). ELISAanalysis: Whereas neither TNF-α and INF-γ in unstimulated PBMC-derivedsupernatants were detected, supernatants from PHA or CD3 stimulatedPBMCs evidenced high values for these cytokines as indicated by ELISAanalysis (**p<0.001, *p<0.05, n=8). The results clearly show a differentsecretion pattern of inflammatory cytokines in comparison tounstimulated PBMC.

Conclusion:

These data indicate that “unstimulated PBMC” evidence a distinctdifferent phenotype (activation marker, cytokine secretion) as comparedto stimulated PBMCs (PHA and CD3 mAb).

FIG. 6A indicates that neither unstimulated viable PBMCs or IA-PBMCssecrete the mainly monocyte derived pro-inflammatory cytokine TNF-α.(Significances are indicated as follows: *p=0.05, **p=0.001; n=8)

FIG. 6B demonstrates a strong induction of pro-inflammatory Interferon-γsecretion after activation as compared to unstimulated PBMC.(Significances are indicated as follows: *p=0.05, **p=0.001; n=8)

FIG. 7A shows pooled results of flow cytometric analysis. PBMCs weregated for T cells and expression of activation markers CD69 and CD25were evaluated. (Significances are indicated as follows: *p=0.05,**p=0.001; n=4)

FIG. 7B displays a representative FACS analysis of PBMCs eitheractivated (PHA, CD3 mAb). Gating represents % of positive cells.

Example 3 Proliferative Activity of PBMCs Cultivated in a PhysiologicalSolution

The aim of this example is to prove that PBMCs have no proliferativeactivity as compared to immune assays that utilize specific (CD3),unspecific (lectin, PHA) and allogeneic T-cell triggering (mixedlymphocyte reaction, MLR) in a 2 day (CD3, PHA) and 5 day (MLR)stimulation assay.

Material and Methods

PBMCs were separated from young healthy volunteers by Ficoll densitygradient centrifugation and resuspended in RPMI (Gibco, USA) containing0.2% gentamycinsulfate (Sigma Chemical Co, USA), 1% L-Glutamin (Sigma,USA) at 1×10⁵ cells per 200 μL. Responder cells were either stimulatedby MoAb to CD3 (10 μg/mL, BD, NJ, USA), PHA (7 μL/mL, Sigma Chemical Co,USA) or with irradiated allogeneic PBMCs at a 1:1 ratio (for MLR).Plates were incubated for 48 h or 5 days and then pulsed for 18 h with3[H]-thymidine (3.7×10⁴ Bq/well; Amersham Pharmacia Biotech, Sweden).Cells were harvested and 3[H]-thymidine incorporation was measured in aliquid scintillation counter.

Results

Stimulated PBMCs showed high proliferation rates as measured by3[H]-thymidine incorporation when compared to viable PBMCs cultured inRPMI without stimulation (FIG. 8). This effect was observed by adding Tcell specific stimuli (PHA, CD3) as well as in assays whereproliferation was triggered by antigen presenting cells (MLR).

Conclusion

This set of experiments implicates that viable PBMCs held in culture forup to 5 days did not proliferate, whereas PBMCs stimulated by differentways showed a marked proliferative response. It is concluded thatculture of PBMCs without stimulation does not lead to proliferativeresponse.

Example 4 Secretoma of Separated PBMCs Kept Under Sterile CultureConditions Possess Neo-Angionetic Capacity

Since neo-angionesis and inflammation are strongly linked in vivo it wasinvestigated whether these secretoma of PBMCs also exhibitanti-proliferative effects on T cells and therefore interfere with aninflammatory immune response.

Material and Methods

Secretoma were obtained by incubating PBMCs (2.5×10⁶/mL) from younghealthy volunteers separated by Ficoll density gradient centrifugationfor 24 h in RPMI (Gibco, Calif., USA) containing 0.2% gentamycinsulfate(Sigma Chemical Co, USA), 1% L-Glutamine (Sigma, USA). Supernatants wereseparated from the cellular fraction and stored at −80° C. Forproliferation assays allogeneic PBMCs were resuspended at 1×10⁵ cellsper 200 μL RPMI after separation. Responder cells were either stimulatedby MoAb to CD3 (10 μg/mL, BD, USA) or PHA (7 μL/mL, Sigma Chemical Co,USA). Different dilutions of supernatants were added. Plates wereincubated for 48 h and then pulsed for 18 h with ³[H]-thymidine (3.7×10⁴Bq/well; Amersham Pharmacia Biotech, Sweden). Cells were harvested and³[H]-thymidine incorporation was measured in a liquid scintillationcounter.

Results:

Secretoma of allogeneic PBMCs evidenced a significant reduction ofproliferation rates measured by ³[H]-thymidine incorporation whencompared to positive controls (FIG. 9). This effect was dose-dependentand could be seen upon anti-CD3 as well as upon PHA stimulation.

Implication:

This set of experiments implicates that secretoma obtained from viablePBMCs held in culture for 24 h exhibit significant anti-proliferativeeffects in vitro. These data indicate that supernatant derived fromPBMCs or in lyophilized form may serve as potential therapeutic formulato treat human diseases that are related to hypoxia induced inflammationor other hyperinflammatory diseases (e.g. auto-immune diseases,inflammatory skin diseases).

Example 5 Paracrine Factors Secreted by Peripheral Blood MononuclearCells Preserve Cardiac Function

In example 1 it was shown that transfusion of cultured irradiatedapoptotic cells derived from peripheral blood significantly improvedfunctional cardiac recovery after experimental myocardial infarction inrats. This improvement was based on immunosuppressive features ofapoptotic cells, pro-angiogenic effects and induction of augmentedhoming of c-kit+ endothelial progenitor cells (EPCs).

In the present example peripheral blood mononuclear cells (PBMC) eitherviable or irradiated with a dose of 60 Gray were incubated for 24 hoursto generate conditioned cell culture supernatants. Supernatants werelyophilized and kept frozen until use in in vivo experiments. Myocardialinfarction was induced in Sprague-Dawley rats by ligating the leftanterior descending artery. After the onset of ischemia, lyophilizedsupernatants were resuspended and injected intravenously. Tissue samplesfor histological and immunohistological evaluations were obtained threedays and six weeks after myocardial infarction. Cardiac function wasassessed by echocardiography six weeks post AMI. Sham operated anduntreated animals served as controls.

Rats that were infused with supernatants obtained from apoptotic PBMCsevidenced increased myocardial angiogenesis and enhanced homing ofendothelial progenitor cells within 72 hours as compared to controls.Planimetric evaluation of fibrotic areas indicated reduced infarctionsize in animals treated with supernatants from apoptotic cells.Furthermore, echocardiography showed a significant improvement regardingpost AMI remodeling as evidenced by an attenuated loss of ejectionfraction and preserved ventricular geometry. Left ventricular ejectionfraction (LVEF) in rats receiving supernatants from apoptotic cellsevidenced a mean value of 56±4% compared to 60±5% in sham operatedanimals, whereas untreated or viable cell supernatant infused animalsshowed a significant decline of LVEF to 44±3% and 41±4% respectively(p<0.001).

These data indicate that infusion of supernatants derived fromirradiated apoptotic PBMCs in experimental AMI circumvented inflammationand caused preferential homing of regenerative EPC leading topreservation of ventricular function.

Methods Cell Culture of Human PBMCs for In Vitro Assays

Human peripheral blood mononuclear cells (PBMC) were obtained by Ficolldensity grade centrifugation as described previously. To induceapoptosis in human PBMC, cells were irradiated with 60 Gy (irradiationautomat for human blood products, Department of Hematology, GeneralHospital Vienna). Both viable and irradiated apoptotic (IA-) PBMCs wereincubated at 37° Celsius for 24 hours at various cell densities (1×10⁶,5×10⁶, 10×10⁶ and 25×10⁶ cells/milliliter, n=5). Then supernatants wereobtained and levels of secreted proteins were measured by Enzyme-linkedimmunosorbent assay (ELISA, R&D Systems, Minneapolis, USA), according tothe protocols supplied by the manufacturer.

Acquisition of Syngeneic IA-PBMCs and Viable-PBMCs for AMI In VivoExperiment

Syngeneic rat PBMCs for in vivo experiments were separated by densitygradient centrifugation from whole-blood obtained from prior heparinizedrats by puncturing of right atrium. Apoptosis was induced by Cs-137cesium irradiation with 45 Gy for in vivo experiments and cultured at37° Celsius at a cell density of 25×10⁶ cells/milliliter). Induction ofapoptosis by irradiation was measured by flow cytometry (annexin Vstaining >80% for IA-PBMC, annexin V staining <20% for viable PBMC).Cells were incubated for 24 hrs in a humidified atmosphere (5% CO2, 37°C., relative humidity 95%). Supernatants were removed and dialyzed witha 3.5 kDa cutoff (Spectrum laboratories, Breda, The Netherlands) against50 mM ammonium acetate overnight at 4° C. Then supernatants were sterilefiltrated and lyophilized. Lyophilized secretoma were stored at −80° C.and freshly resuspended for every experiment. Secretoma were randomsampled for their pH value. The lyophilized powder was stored at −80°Celsius until further experiments were conducted.

Induction of Myocardial Infarction

Animal experiments were approved by the committee for animal research,Medical University of Vienna. All experiments were performed inaccordance to the Guide for the Care and Use of Laboratory Animals bythe National Institutes of Health (NTH). Myocardial infarction wasinduced in adult male Sprague-Dawley rats by ligating the left anteriordescending artery (LAD). In short, animals were anesthetizedintraperitoneally with a mixture of xylazin (1 mg/100 g bodyweight) andketamin (10 mg/100 g bodyweight) and ventilated mechanically. A leftlateral thoracotomy was performed and a ligature using 6-0 prolene wasplaced around the LAD beneath the left atrium. Immediately after theonset of ischemia, lyophilized supernatants obtained from 8×10⁶apoptotic PBMCs resuspended in 0.3 mL cell culture medium were infusedover the femoral vein. Infusion of cell culture medium alone, viablePBMC supernatants and sham operation served as negative controls in thisexperimental setting, respectively. The experimental design is shown inFIG. 1.

Histology and Immunohistochemistry In Vivo

See example 1.

Determination of Myocardial Infarction Size by Planimetry

See example 1.

Cardiac Function Assessment by Echocardiography

See example 1.

Statistical Methods

Statistical analysis was performed using Graph Pad Prism software (USA).All data are given as mean±standard error of the mean. Paired two-sidedt-tests for dependent, unpaired t-tests for independent variables wereutilized calculating significances.

Between-group differences regarding survival of acute myocardialinfarction were compared by Kaplan-Meier actuarial analysis.Bonferroni-Holm correction was used to adjust p-values for multipletesting. P-values <0.05 were considered statistically significant.

Results Determination of Paracrine Factors Secreted by IA-PBMCs andViable PBMCs by ELISA

The results are shown in FIGS. 10 to 15.

Example 6 Paracrine Factors Secreted by Peripheral Blood MononuclearCells Posses Immunosuppressive Features

In Example 1, anti-inflammatory effects of PBMC secretoma in an acutemyocardial infarction (AMI) animal model are evidenced. In this exampleit is shown that the application of PBMC secretoma after AMI inductioninhibits the inflammatory damage of the heart muscle by massivelydown-regulating the immune response.

Based on these findings possible immunosuppressive effects of secretomain in vitro experiments were investigated. CD4+ cells play a key role inthe orchestration of the immune response as they are pivotal for theassistance of other leukocytes (e.g. macrophages, B cells, cytotoxic Tcells) in immunological processes.

Material and Methods Production of PBMC Secretoma

PBMCs from healthy volunteers were separated by Ficoll densitycentrifugation. Cells were resuspended in Ultra Culture Medium (Lonza,Basel, Switzerland) at a concentration of 1×10⁶ cells/mL (sup liv). Forthe production of secretoma from apoptotic PBMCs was induced byirradiation with 60 Gy (sup APA). Cells were incubated for 24 h in ahumidified atmosphere (5% CO2, 37° C., relative humidity 95%).Supernatants were removed and dialyzed with a 3.5 kDa cutoff (Spectrumlaboratories, Breda, The Netherlands) against 50 mM ammonium acetateovernight at 4° C. Then supernatants were sterile filtrated andlyophilized. Lyophilized secretoma were stored at −80° C. and freshlyresuspended for every experiment. Secretoma were random sampled fortheir pH value.

Separation of CD4 Cells

CD4+ cells were separated by depletion of non-CD4+ T cells utilizing aMACS bead system (Miltenyi, Bergisch Gladbach, Germany). Cells werefreshly prepared and immediately used for each experiment.

Measurement of Apoptosis

Apoptosis was detected by flow cytometry using a commercially availableAnnexin V/PI kit (BD, New Jersey, USA). Apoptosis were defined byAnnexin positive staining, late apoptosis by PI positivity.

Proliferation Experiments

PBMCs or purified CD4+ cells were diluted in Ultra Culture supplementedwith 0.2% gentamycinsulfate (Sigma, St. Louis, Mo., USA), 0.5%β-mercapto-ethanol (Sigma, St Louis, Mo., USA) and 1% GlutaMAX-I(Invitrogen, Carlsbad, Calif., USA) to a concentration of 1×10⁵/well ina 96 round-bottom well plate. Cells were stimulated with either PHA (7μg/mL, Sigma, USA), CD3 (10 μg/mL, BD, New Jersey, USA) IL-2 (10 U/mL,BD, USA) or a 1:1 ratio of allogeneic irradiated (60 Gy) PBMCs for MLR.Cells were incubated for 48 h or 5 days (MLR) with differentconcentrations of PBMC secretoma, IL-10 or TGF-β. Then cells were pulsedfor 18 h with 3[H]-thymidine (3.7×10⁴ Bq/well; Amersham PharmaciaBiotech, Uppsala, Sweden). Cells were harvested and 3[H]-thymidineincorporation was measured in a liquid scintillation counter.

Activation Markers

Purified CD4+ cells were stimulated with anti-CD3 (10 μg/mL) andco-incubated with different concentration of PBMC secretoma. Cells werestained for CD69 and CD25 following a standard flow cytometric stainingprotocol and analyzed on a flow cytometer FC500 (Beckman Coulter,Fullerton, Calif., USA).

Results

In preliminary experiments the anti-proliferative properties of PBMCsupernatants from viable cells (sup liv) were tested. In anti-CD3 andPHA stimulation experiments proliferations rates were significantlyreduced by the addition of secretoma (n=10).

Based on these findings the effect of PBMC secretoma on the T-helpercell compartment was evaluated, since these cells play a pivotal role inlaunching and perpetuating an immune response. In analogy to FIG. 16highly purified CD4-cells lost their proliferative capacity by theaddition of secretoma. This phenomenon was observed for the supernatantof living as well as of apoptotic, irradiated PBMCs (FIG. 17, n=5).

The next step was to determine possible effects of the secretoma on cellviability. Therefore, resting CD4+ cells were incubated with supernatantand Annexin V and PI positivity was evaluated. Supernatants from both,living and apoptotic PBMC, evidenced remarkable pro-apoptotic effects(FIG. 18, n=5).

To test if PBMC secretoma were able to inhibit CD4+ cell activation theT cell activation markers CD25 and CD69 following anti-CD3 stimulationof CD4+ cells was evaluated. The upregulation of both markers wassignificantly and dose-dependent inhibited by PBMC secretoma (FIG. 19,n=5).

In a last set of experiments the effect of the immune-suppressivecytokines IL-10 and TGF-β by the addition of neutralizing antibodies inthese experiments was examined. Neither IL-10 and TGF-β was found to beresponsible for the anti-proliferative effects of our PBMC secretoma,since demonetizing these cytokines did not increase proliferation rates(FIG. 20, n=5).

CONCLUSION

These experiments evidence for the first time that PBMC secretoma possesimmune-suppressive features in vitro. It was shown that supernatant a)reduces proliferation rates in anti-CD3, PHA and MLR stimulationexperiments, b) has the potency to induce apoptosis and inhibitsactivation of CD4+ cells upon T cell triggering.

1-16. (canceled)
 17. A method of treating an inflammatory condition, comprising: administering a pharmaceutical preparation comprising a cell-free culture supernatant of peripheral blood mononuclear cells (PBMCs) to a patient in need of treatment for an inflammatory condition selected from the group consisting of myocardial ischemia, limb ischemia, tissue ischemia, ischemia-reperfusion injury, angina pectoris, coronary artery disease, peripheral vascular disease, peripheral arterial disease, stroke, ischemic stroke, myocardial infarct, congestive heart failure, trauma, bowel disease, mesenterial infarction, pulmonary infarction, bone fracture, tissue regeneration after dental grafting, auto-immune diseases, rheumatic diseases, transplantation allograft and rejection of allograft, the PBMCs comprising non-activated, non-proliferating T cells, B cells, NK cells, and monocytes cultivated under a stress inducing condition in a physiological solution free of PBMC-proliferating and PBMC-activating substances for at least 1 h.
 18. The method of claim 17, wherein the cell-free culture supernatant comprises at least 2000 pg/mL of matrix metallopeptidase 9 (MMP-9) and no detectable amount of TNF-α.
 19. The method of claim 17, wherein the cell-free culture supernatant has no detectable amount of INF-γ.
 20. The method of claim 17, wherein the stress inducing condition comprises a dose of γ-radiation.
 21. The method of claim 20, wherein the dose of γ-radiation is at least 10 Gy.
 22. The method of claim 20, wherein the dose of γ-radiation is at least 20 Gy.
 23. The method of claim 20, wherein the dose of γ-radiation is at least 40 Gy.
 24. The method of claim 17, wherein the pharmaceutical preparation is administered by subcutaneous administration, intramuscular administration, intra-organ administration, or intravenous administration.
 25. The method of claim 17, wherein the physiological solution is selected from the group consisting of a salt solution, whole blood, blood fraction, and a cell culture medium.
 26. The method of claim 23, wherein the salt solution is a physiological NaCl solution.
 27. The method of claim 17, wherein the blood fraction is serum.
 28. The method of claim 17, wherein the PBMCs are cultivated in the physiological solution for at least 4 hours.
 29. The method of claim 17, wherein the PBMCs are cultivated in the physiological solution for at least 12 hours.
 30. The method of claim 17, wherein the culture supernatant comprises PBMCs.
 31. A method of treating an inflammatory condition, comprising: administering a pharmaceutical preparation comprising a cell-free culture supernatant of peripheral blood mononuclear cells (PBMCs) to a patient in need of treatment for an inflammatory condition selected from the group consisting of myocardial ischemia, myocardial infarct, and congestive heart failure, the PBMCs comprising non-activated, non-proliferating T cells, B cells, NK cells, and monocytes cultivated under a stress inducing condition in a physiological solution free of PBMC-proliferating and PBMC-activating substances for at least 1 h.
 32. The method of claim 31, wherein the cell-free culture supernatant comprises at least 2000 pg/mL of matrix metallopeptidase 9 (MMP-9), no detectable amount of TNF-α, and no detectable amount of INF-γ.
 33. The method of claim 31, wherein the stress inducing condition comprises a dose of at least 10 Gy radiation.
 34. The method of claim 31, wherein the pharmaceutical preparation is administered by intra-organ administration or intravenous administration.
 35. A method of treating an inflammatory condition, comprising: administering a pharmaceutical preparation comprising a cell-free culture supernatant of peripheral blood mononuclear cells (PBMCs) to a patient in need of treatment for an inflammatory condition selected from the group consisting of myocardial ischemia, myocardial infarct, and congestive heart failure, the cell-free culture supernatant comprising at least 2000 pg/mL of matrix metallopeptidase 9 (MMP-9) and no detectable amount of TNF-α, the PBMCs comprising non-activated, non-proliferating T cells, B cells, NK cells, and monocytes cultivated under a stress inducing condition comprising a dose of at least 10 Gy radiation in a physiological solution free of PBMC-proliferating and PBMC-activating substances for at least 1 h.
 36. The method of claim 35, wherein the pharmaceutical preparation is administered by intra-organ administration or intravenous administration. 